Preliminary Studies On Gene Editing Of BnTT12 And Mapping Of Yellowish-White Flower Trait Locus In Brassica Napus

Breeding of yellow-seeded oilseed rape with high oil content in the seeds,good quality of oil is an objective in the genetics and breeding.The research on oilseed rape flower colour as an indicator trait in breeding and as an ornamental trait is also of significance.Present study is on gene editing of BnTT12 related to seed oil content and mapping of a yellowish-white locus in Brassica napus.The main results are as follows:1.Editing of Bn TT12 gene（1）Cloning of BnTT12 gene:By querying the B.napus genome database,gene-specific primers for transparent testa gene BnTT12 were designed,then BnTT12-1 and BnTT12-2 were cloned.（2）Subcellular localization:The fusion expression vectors containing BnTT12-1 and BnTT12-2 gene and GFP were constructed,respectively,and injected tobacco.By observation with laser confocal microscopy on tabacco leaves,BnTT12-1 and BnTT12-2 genes were demonstrated expressed in cell membranes.（3）Editin of BnTT12:A target site of gene editing was designed in the conserved domain of BnTT12-1 and BnTT12-2.Atu3d-gRNA promoter expression box was used to construct expression box containing target sequence,and then connected to editing vector pYLCRISPR/Cas9p35S-H to form a complete editing vector,which was used to knock out BnTT12 gene.Agrobacterium-mediated transformation was used to transform the rapeseed variety of ZS11.Six transgenic strains were screened and the grain color turned yellow at the maturity stage.2.Mapping of an yellowish-white flower locus（1）Inheritance of the yellowish-white flower trait:Yellowish-white flower NJAU5738 was crossed reciprocally with Zhongshuang 11（ZS11）.generating F₁ plants with yellowish-white flowers（YWF）.This indicated that the YWF trait is dominant.The number of yellowish-white flower and yellow flower correspond to 3:1 in F₂ generation.The selfing progeny of the cross between NJAU5738 and ZS11 consistently indicated that the YWF trait is controlled by a pair of dominant allel.The site was named BnYWF.（2）Mapping of the BnYWF:Using the advanced generation derived from NJAU5738 and ZS11,candidate chromosomal segments locating BnYWF locus were found by aid of SNP genotyping with Brassica 60K SNP Beadchip Array.Then,SSR primers were designed based on B.napus genome sequence.17 pairs of primers were found polymorphic between the two parents.Therafter,advanced backcross population was screened with these SSR markers.At last,BnYWF was located next to SSR marker BnaC05-274 on C05 chromosome with a distance of 4.8cM.